Complement (C) is a key arm of innate immune defenses against invasive meningococcal infections. Neisseria meningitidis (Nm) have evolved several sophisticated mechanisms to evade host C. A balance between the hosts' efforts to activate C on the bacterium and the microbe's C evasion strategies dictates whether the organism is cleared from the mucosa, remains an asymptomatic colonizer in the nasopharynx, or proceeds to cause invasive disease. In the previous funding period we characterized interactions between Nm and C4b, a component of the classical pathway (CP). Almost all Nm isolates recovered from the blood or cerebrospinal fluid are encapsulated and capsule is important for resistance to C. The molecular basis for capsule-mediated C resistance remains undefined. In Aim 1 will define the role of capsular polysaccharide (CPS) in regulating C. First, in Aim 1a, we will elucidate the reason for differences in the level of CP activation mediated by anti-CPS and anti-outer membrane Abs. On a molar basis, mAbs directed against CPS fix less C4b than mAbs against membrane structures, suggesting that efficient C4b binding may require activation of C4 proximate to the meningococcal molecules that serve as C4b acceptors such as LOS and opacity protein. Chimeric mAbs containing human IgG1 Fc and directed against either CPS or the Nm vaccine candidate, factor H-binding protein (fHbp) will be used to symmetrically compare the C-activating functions of these mAbs. The positive feedback loop of the alternative pathway (AP) is also important for bacterial killing by vaccine Abs. Our preliminary studies indicate that the group A, C, W-135 and Y (but not B) CPSs block activation of human C3 by purified human factors B and D. In Aim 1b we will define the molecular basis of AP inhibition by examining the interaction of CPS with purified AP components. These studies may identify a common mechanism of action for polyanions that block AP activation. AP regulation by CPS is human-specific; assembly of the rabbit AP (not regulated by CPS) will be examined in Aim 1c to provide a better understanding of the human-specificity of AP regulation by CPS. Surface proteins also contribute to C resistance; fHbp binds to the AP inhibitor, factor H (fH) and inhibits C activation. In Aim 2, we will we will exploit our knowledge of fH- fHbp interactions and use chimeric molecules to block fH binding to Nm and manipulate the C cascade to precisely define the roles of the CP and AP in killing Nm. We will also attempt to 'boost' the bactericidal function of an otherwise nonbactericidal anti-fHbp mAb (JAR 4). Such knowledge could lead to optimization of vaccine strategies. Finally, in Aim 3 we will define novel C evasion strategies in strains that express low levels of fHbp. Preliminary evidence indicates that low fHbp expressing/low fH binding Nm regulate C at the level of C5b-9 formation or insertion and we will characterize the molecular basis for C resistance at this level. These studies will help define novel mechanisms of C evasion by Nm; information that will advance understanding of meningococcal pathogenesis and improve ongoing efforts to develop effective protein-based vaccines. PUBLIC HEALTH RELEVANCE: Meningococcal meningitis and sepsis is a major health problem worldwide. It mainly affects children and young adults and is a significant cause of morbidity and mortality. Although a good vaccine exists against several meningococcal serogroups, there is no effective vaccine against serogroup B disease. The emergence of disease caused by strains that lack a capsule following vaccination campaigns using capsular polysaccharide-based vaccines is of concern. Complement forms an important arm of host defenses against the meningococcus. The studies proposed in this application will help us better understand how this pathogen escapes killing by complement and will ultimately aid in the development of better vaccines against meningococcal meningitis.